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Histone Methylation by Temozolomide; a Classic DNA Methylating Anticancer Drug

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Histone Methylation by Temozolomide; a Classic DNA Methylating Anticancer Drug ANTICANCER RESEARCH 36: 3289-3300 (2016) Histone Methylation by Temozolomide; A Classic DNA Methylating Anticancer Drug TIELI WANG1, AMANDA J. PICKARD2 and JAMES M. GALLO2 1Department of Chemistry and Biochemistry, California State University Dominguez Hills, Carson, CA, U.S.A.; 2Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY, U.S.A. Abstract. Background/Aim: The alkylating agent, adults (1). Despite advances in multimodal therapies of temozolomide (TMZ), is considered the standard-of-care for surgical extirpation, radiotherapy and chemotherapy, GBMs high-grade astrocytomas –known as glioblastoma multiforme remain a cancer of poor prognosis that can be attributed to (GBM)– an aggressive type of tumor with poor prognosis. The the aggressive nature of the tumors and the development of therapeutic benefit of TMZ is attributed to formation of DNA resistance to radiation and chemotherapy (2). Out of adducts involving the methylation of purine bases in DNA. We chemotherapeutic approaches to GBMs, temozolomide investigated the effects of TMZ on arginine and lysine amino (TMZ) is the main alkylating agent used, based on its ability acids, histone H3 peptides and histone H3 proteins. Materials to increase the median survival (3-6). and Methods: Chemical modification of amino acids, histone TMZ possesses favorable pharmacokinetic characteristics H3 peptide and protein by TMZ was performed in phosphate with high oral bioavailability and blood-brain barrier (BBB) buffer at physiological pH. The reaction products were penetration (7). TMZ is a prodrug that undergoes pH- examined by mass spectrometry and western blot analysis. dependent degradation, first producing 5-(3-methyltriazen-1- Results: Our results showed that TMZ following conversion to yl)imidazole-4-carboxamide (MTIC), that is subsequently a methylating cation, can methylate histone H3 peptide and degraded into 4-amino-5-immidazole-carboxamide (AIC) – an histone H3 protein, suggesting that TMZ exerts its anticancer inactive metabolite and a methyldiazonium cation, the activity not only through its interaction with DNA, but also methylating agent (Figure 1). The anticancer effects of TMZ through alterations of protein post-translational modifications. are attributed to the methylation of DNA by TMZ with the Conclusion: The possibility that TMZ can methylate histones primary cytotoxic lesion being O6-methylguanine (O6-MeG); involved with epigenetic regulation of protein indicates a N7-methylguanine, N3-methylguanine, and N3-methyladenine potentially unique mechanism of action. The study will (8-10) adducts also contribute to the cytotoxicity. The DNA contribute to the understanding the anticancer activity of TMZ adducts can lead to single- and double-strand DNA breaks and in order to develop novel targeted molecular strategies to eventually apoptosis; however, numerous DNA repair systems, advance the cancer treatment. highlighted by the methylguanine methyltransferase (MGMT) enzyme that repairs the O6-MeG lesions, can result in Malignant gliomas, or glioblastoma multiforme (GBM), are chemoresistance (11-13). Other DNA repair processes, such as the most common primary brain malignancies found in mismatch repair (MMR) and base excision repair (BER), also influence cellular resistance to TMZ. The field of epigenetics characterizes how chemical and enzymatic modifications to DNA and histones occur and This article is freely accessible online. their subsequent impact on gene expression. In fact, Correspondence to: Tieli Wang, Department of Chemistry and hypermethylation of the MGMT promoter in GBM patients Biochemistry, California State University Dominguez Hills, Carson, (14, 15, 16) is used as a favorable prognostic biomarker and CA, U.S.A. Tel: 310 2433388, Fax: 310 2432593, e-mail: increases drug sensitivity since a lower amount of the [email protected] and James M. Gallo, Department of enzyme is available to repair TMZ-induced lesions (17). Pharmacology and Systems Therapeutics, Icahn School of Medicine Although DNA methylation has been shown to affect at Mount Sinai, New York, NY, U.S.A. Tel: 212 2413980, Fax: 212 chromatin structure and influence histone methylation (18), it 9967214, e-mail: [email protected]. is direct enzymatic modification of histones, such as Key Words: Temozolomide, malignant gliomas, chemotherapy, methylation and acetylation that are considered crucial to histone methylation. gene expression. 0250-7005/2016 $2.00+.40 3289 ANTICANCER RESEARCH 36: 3289-3300 (2016) Figure 1. TMZ metabolic cascade at physiological pH to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) and its metabolites, 4-amino-5- imidazole-carboxamide (AIC) and methyldiazonium ion. In the current study, we addressed whether TMZ can reaction mixture was centrifuged in an Amicon Ultra-0.5 centrifugal methylate histones directly, by examining the effects of TMZ filter device (EMD Millipore, Billerica, MA, USA) at 4˚C at 14,000 × on amino acids, peptides and histone proteins in vitro using g with a 40˚ fixed angle rotor to concentrate the protein samples and remove low molecular weight impurities and unreacted TMZ. The 100 mass spectrometry and protein biochemical methods. Our μg concentrated protein solution was resuspended in 100 μl 0.01 M results demonstrated that TMZ is able to methylate histone phosphate buffer (pH 7.4). Western blot analysis was then used to proteins under physiological conditions. The possibility that confirm the methylated histone H3 protein. this action could occur in GBM patients opens a new avenue of investigation to determine its significance. Mass spectrometry. Mass spectrometry experiments were performed using a QTRAP 5500 triple quadrupole mass spectrometer (AB Materials and Methods SCIEX, Framingham, MA, USA) coupled to a Shimadzu Prominence UFLC system (Shimadzu Corp., Torrance, CA, USA). The system was continuously supplied with ultra-high purity Chemicals and general procedures. Arginine, lysine and TMZ nitrogen, dry air, and zero air by a Source 5000 LC-MS TriGas (>98%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, generator (Parker Hannifin Corp., Haverhill, MA, USA). The USA). Histone H3 (1-8) peptide H2N-ARTKQTAR-COOH (>95% QTRAP 5500 system was equipped with an electrospray ionization by HPLC, MW=931.1 g/mol) was purchased from AnaSpec, Inc. (ESI) TurboIon Spray probe utilizing zero grade air as the nebulizer (Fremont, CA, USA). Xenopus recombinant histone H3 (C110A) (Gas 1) and heater (Gas 2) gases. Ultra-high purity nitrogen was (>98% by SDS-PAGE, MW=15,239 Da), supplied as a lyophilized used as the curtain (CUR) and collision (CAD) gases. Mass powder, was purchased from Active Motif (Carlsbad, CA, USA). spectrometer control and spectral processing were carried out using Recombinant human histone H3.1 protein (MW=15273.2 Da by Analyst 1.5.2 software (AB SCIEX). Mass spectrometer parameters ESI-TOF) was purchased from New England BioLabs, Inc. were optimized for each compound analyzed (Tables I and II). A (Ipswich, MA, USA). Human recombinant histone H3 protein 1μl sample of unreacted amino acid, TMZ, or the reaction mixture (≥95% by SDS-PAGE, MW=15,500 Da by SDS-PAGE) was was introduced into the electrospray source by direct injection. The purchased from Cayman Chemical Co. (Ann Arbor, MI, USA) as a mobile phase consisted of 50% methanol with 0.1% formic acid and lyophilized powder. All buffers and mobile phases were prepared 50% water with 0.1% formic acid and was maintained at a constant using analytical grade reagents and ultrapure water from a Millipore flow rate of 200 μl/min. Unreacted and reacted peptide solutions Synergy UV water filtration system (EMD Millipore, Billerica, MA, were infused directly into the electrospray source at a flow rate of 7 USA) at a resistivity of 18 MΩ·cm. All reagents were used without μl/min. All mass spectra were acquired in positive-ion mode with further purification. The pH measurements were measured using a unit mass resolution. Mass spectra for the free amino acid studies calibrated Fisher Scientific AccuMet Basic AB15 pH MV benchtop were background-subtracted with a spectrum obtained of 100 μM pH meter (Fisher Scientific, Pittsburgh, PA, USA). TMZ in 50/50 0.01 M phosphate buffer/methanol (v/v). Peptides were fragmented under conditions with the collision Reactivity of amino acids and peptides with TMZ. Separate stock energy between 15 to 45 eV by the direct infusion method at a flow solutions of arginine (0.5 mM), lysine (0.5 mM), TMZ (0.2 mM) rate of 7 μl/min. The MS/MS spectra were interpreted manually, and histone H3 (1-8) peptide (10 mg/ml) were prepared in 0.01 M primarily by assigning the peaks corresponding to the C-terminal y- phosphate buffer (pH 7.4) and kept at 4˚C until use. A solution ions. The modification was mapped based on the mass difference of containing 10 μM amino acid or peptide and 200 μM TMZ was the y ions in the spectrum derived from the doubly charged mixed at room temperature for 24 h. Subsequently an aliquot of the precursor ions when compared to the y ions in the spectrum of solution was diluted 1:1 with methanol and was passed through a unmethylated peptide. Mass spectrometer parameters were 0.22 μm syringe filter. The filtered solution was subsequently optimized for each methylated peptide analyzed (Table III). Mass analyzed by LC-MS/MS. fragmentation prediction was generated using Mascot (Matrix Science, London, UK). Reactivity of histone H3 Proteins with TMZ. 100 μg of histone H3 Western blot analysis. Standard protein electrophoresis conditions and protein was resuspended in 100 μl 0.01 M phosphate buffer (pH 7.4) reagents were used for gel and sample preparation. Recombinant and 10 μM TMZ in 0.01 M (pH 7.4) phosphate buffer was mixed with proteins (0.5-5 μg) and the LI-COR Chameleon Duo ladder (LI-COR the protein solution at room temperature for 24 h. The resultant Biosciences, Inc., Lincoln, NE) were loaded onto a 12% Mini-Protean- 3290 Wang et al: Protein Methylation by Temozolomide Table I.
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